Anthrax is an infection caused by the spore-forming bacterium Bacillus anthracis. Anthrax may enter the body and cause infection by means of inhalation, ingestion or subcutaneous exposure. While animals are most at risk for anthrax exposure, humans working with such animals may also be at risk. Additionally, recent heightened awareness of the possibility of bioterrorism has raised concerns about the use of B. anthracis or related strains, both newly emerging or genetically engineered, as bio-weapons.
There is therefore a need to develop vaccines for widespread use in the event of a bioterrorist attack, in order to minimize the exposure of a population to the bacteria. In particular, the ability to confer protection following oral dosing is particularly attractive in the context of a bioterrorism event as it would greatly simplify the process of mass vaccinations. Additionally, such a vaccine may be used in situations where a population may be at high risk of exposure to the bacteria, whether through bioterroristic activity or natural exposure, due to proximity to infected animals or to the spores.
The infective process of anthrax occurs when the spores are taken up by the body, through inhalation, ingestion or subcutaneous exposure. The spores become active toxic bacteria and express anthrax toxin, which will ultimately halt the host's immune response and cause cell death. Anthrax toxin has three components: anthrax protective antigen (PA), anthrax edema factor (EF) and anthrax lethal factor (LF). PA binds an anthrax toxin receptor (ATR) on the surface of the host cell. The PA is then cleaved by a host protease, activating the PA, which then binds to other active PAs to form a heptamer. The heptamer then binds EF or LF and the entire complex is drawn into the cell via endocytosis, forming an endosome within the host cell. The EF or LF is ejected from the endosome, into the cytosol of the cell. Once in the cytosol, LF and EF exert their enzymatic activities, interrupt cell signaling and damage the cells. EF ultimately causes edema and LF ultimately causes cell lysis.
The current FDA approved vaccine is a sterile product made from an avirulent, nonencapsulated strain of B. anthracis. The vaccine was approved by the FDA in 1970 and is generally administered to those considered at high risk, especially those in the United States who work in close contact with potentially infected animals or animal products, such as hides, hairs or bones, e.g. veterinarians and laboratory workers. The vaccine, BioThrax® (Anthrax Vaccine Adsorbed or “AVA”), is manufactured by one company, Emergent Biosolutions of Gaithersburg, Md. (formerly Bioport Corporation, Lansing, Mich.). Possible reactions to the vaccine include local reactions, and very rare systemic reactions, causing flu-like symptoms. This vaccine requires six vaccinations over eighteen months (at 0, 2 and 4 weeks and at 6, 12 and 18 months), followed by yearly boosters; see BioThrax® AVA, prescription information, dosage instructions.
Various additional vaccines have been developed against anthrax. PA, as a potent immunogen is generally the target for such vaccines. PA is non-toxic and has been shown in numerous animal studies to be capable of stimulating the production of protective antibodies when given as a vaccine. It is thought that these antibodies protect by inhibiting the binding of PA to the host cell and/or binding to EF and/or LF. However, current vaccines suffer from problems such as poor levels of expression and the need for multiple dosing.
Thus, while certain prevention and treatment approaches may prove useful in modulating the effects of anthrax toxin, there remains a need for an effective and safe vaccine that would effectively produce immunity to anthrax with fewer doses.
Various vaccines have been discussed that target the natural mechanism of PA, LF and/or EF. For example, U.S. Pat. No. 5,591,631 and U.S. Pat. No. 5,677,274 describe fusion proteins including domains of PA and/or LF.
In another approach, U.S. Patent Application No. 2004/0166120 has described a composition which contains PA and a truncated, non-functional B. anthracis LFn for eliciting a B. anthracis immune response.
Additionally, U.S. Patent Application No. 2003/0003109 discusses vaccines that administer a polynucleotide with a coding sequence for a mutated LF protein or an immunogenic fragment of an LF protein and a polynucleotide with a coding sequence for PA or an immunogenic fragment of PA to a subject.
U.S. Patent Application No. 2005/0063986 discusses recombinant DNA constructs containing wild type or mutant type PA, LF or EF.
Additional approaches have focused on live vaccines as expression systems for PA, LF or EF, but have not been able to develop these vaccines for human use. Specific attempts focused on use of live Salmonella (Coulson, et al. Vaccine, vol. 12, No. 15, 1395-1401 (1994); Garmory, et al. Infect. Immun., 71(7): 3831-6 (2003)) and B. anthracis (Aloni-Grinstein, et al. Infect. Immun., 73(7): 4043-53 (2005)) have met with limited success, but have not been developed for human use. Additional work has focused on the possibilities of development of live vaccines (U.S. Application No. 2004/0197343), but have not identified a specific vaccine for use in humans utilizing a live virus containing genetic material from B. anthracis. 
The utility of attenuated strains of Salmonella as a live oral vaccine for typhoid has resulted in the development of a licensed, FDA approved vaccine. There is considerable interest in building on this approach to develop Salmonella based vaccines capable of conferring protection against a range of infectious agents and cancer. In particular, development of a live oral anthrax vaccine would be desirable. Additionally, development of a live oral anthrax vaccine with additional activity against one or more additional pathogens would be desirable. However, attempts to use these methods with regard to anthrax have not succeeded to date. Investigations of the use of a live vaccine for expression and delivery of PA, LF and/or EF, have met with limited success, suffering the problem of low levels of expression. Live vaccines evoke the most effective immunity and are the least expensive to produce but in practice are very difficult to make. Additionally, there is a concern that such vaccines would not be effective against genetically modified strains of B. anthracis or against other strains such as Bacillus cereus G9241, which has acquired the B. anthracis toxins and causes an anthrax-like infection in humans.
Therefore, there remains a need in the art for development of a vaccine using specific antigens from anthrax that are expressed in high quantity and do not require excessive dosing. A live vaccine would be preferred. Such a vaccine would preferably be effective against B. anthracis, genetically modified B. anthracis, anthrax-like strains, and/or additional pathogens, such as plague. In particular, an oral vaccine would be desirable for ease of administration. Such a vaccine is desirable for use in humans.